We propose to continue our program to define biochemically, genetically and structurally the mechanisms by which the viral encoded E1 and E2 proteins of the oncogenic Papillomavirures (PV) work to maintain stable plasmids in dividing cells. E2 is the central regulatory protein in the PV life cycle and is known to control viral DNA replication, transcription and viral DNA tethering to cellular chromosomes. E2 targets a protomer of the E1 protein to the viral DNA replication origin, protein:protein interaction is critical for this targeting function. Subsequently, E1 further assembles into an active and processive DNA helicase. The assembled helicase also interacts with and coordinates the activities of the cellular replication proteins DNA polymerase alpha and the single strand DNA binding protein, RPA. We solved the crystal structure (at 2.1 A ) for the intact HPV-18 activation domain in complex with the helicase domain of E1. The structure suggests new ideas for how the pathway towards a pre-initiation complex proceeds. Aims related to these themes are summarized below. a) We will create E1 mutations in a new "sensor" loop. Such mutations should interfere with E1:E2 interactions but some may not disturb other activities of the initiator protein. b) The X-ray coordinates will be used to create cysteine substitutions in both E1 and E2 and such mutant proteins can be cross-linked by mild oxidation. Our model predicts that disulphide bonds formed at specific locations will profoundly inhibit ATP hydrolysis. c) We will attempt to crystallize different forms of E1 bound to ATP. We propose to create mutants of E1 that will bind but not hydrolyze ATP. Various DNA molecules will be synthesized that might freeze double hexamers in unique conformations on the helicase substrates. Such mutants and substrates will be used for crystallization trials. 2. We will use site directed mutagenesis to employ a structure-based analysis of E1 function. Which surface residues of E1 contribute most to interaction with E2, does the DNA melting step which precedes helicase activity require more than multimerization of the helicase domains? Which surfaces of E1 interact with Polymerase and RPA? 3. Small molecule inhibitors of the E1:E2 complex have been discovered and may prove useful as antiviral agents.These molecules show very distinct viral type specificity. We propose to explore the structural basis for such specificity by creation of specific E2 mutations in one case and to crystallize a drug: E2 complex in another example. 4. We will test the notion that E1 interferes with E2 binding to the cellular Brd4 protein. The latter is thought to be the chromosomal receptor for the viral hitchhiking process. Experiments are also proposed to explore the activities of the HPV-18 E2 protein in this process.